Combination therapy using c-met inhibitor and beta-catenin inhibitor

ABSTRACT

A method of preventing and/or treating a cancer including co-administering a c-Met inhibitor and a beta-catenin inhibitor to a subject in need thereof, a pharmaceutical composition comprising a c-Met inhibitor and a beta-catenin inhibitor, and a kit comprising a pharmaceutical composition comprising a c-Met inhibitor, and a pharmaceutical composition comprising a beta-catenin inhibitor packaged together.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2013-0107568 filed on Sep. 6, 2013 in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: 151,438 bytes ASCII (Text) file named “718342_ST25.TXT,” created Sep. 5, 2014.

BACKGROUND

1. Field

Provided is a method of preventing and/or treating a cancer, the method including co-administering a c-Met inhibitor and a beta-catenin inhibitor to a subject in need thereof

2. Description of the Related Art

It has been shown that resistance to a drug having a specific target is more common compared to resistance to a drug having no specific target. In addition, it is also known that the indications on which a targeting drug has a therapeutic effect when it is treated alone are limited. Co-administration of two or more targeting drugs can lead to a subject overcoming resistance caused by exclusive treatment with only one of the targeting drugs, and can exhibit a therapeutic effect even on an indication on which only one of the targeting drugs have no therapeutic effect, maximizing the therapeutic effect. Such co-administration is expected to contribute to extending the scope of indications to be treated by target drugs and to overcoming resistance thereto.

Therefore, for more effective treatment of a disease, there remains a need to develop effective combination therapy targeting two or more targets.

SUMMARY

The present disclosure relates to a combination therapy targeting c-Met and beta-catenin.

One embodiment provides a pharmaceutical composition for combination therapy including a c-Met inhibitor and a beta-catenin inhibitor as active ingredients.

Another embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating a cancer including a c-Met inhibitor and a beta-catenin inhibitor as an active ingredient.

Another embodiment provides a kit for preventing and/or treating a cancer including a first pharmaceutical composition including a pharmaceutically effective amount of a c-Met inhibitor as an active ingredient, a second pharmaceutical composition including a pharmaceutically effective amount of a beta-catenin inhibitor as an active ingredient, and a package container.

Another embodiment provides a method of preventing and/or treating a cancer including co-administering a c-Met inhibitor and a beta-catenin inhibitor to a subject in need of preventing and/or treating a cancer.

Another embodiment provides a pharmaceutical composition for improving an efficacy of a c-Met inhibitor including a beta-catenin inhibitor.

Another embodiment provides a method of improving an efficacy of a c-Met inhibitor including administering a beta-catenin inhibitor together with a c-Met inhibitor.

Another embodiment provides a pharmaceutical composition for improving an efficacy of a beta-catenin inhibitor including a c-Met inhibitor.

Another embodiment provides a method of improving an efficacy of beta-catenin inhibitor including administering a c-Met inhibitor together with a beta-catenin inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing cell viability of HCC1954 breast cancer cell line when co-treated with L3-1Y/IgG2 and XAV-939.

FIG. 2 is a graph showing cell viability of HCC1954 breast cancer cell line when co-treated with L3-1Y/IgG2 and imatinib.

FIG. 3A is a graph showing cell viability of HT29 colon cancer cell line when co-treated with L3-1Y/IgG2 and XAV-939 (upper portion) and when anti-c-Met antibody 1 and XAV-939 (lower portion).

FIG. 3B is a graph showing cell viability of HT29 colon cancer cell line when co-treated with L3-1Y/IgG2 and imatinib or treated with L3-1Y/IgG2 or imatinib alone.

FIG. 4 is a graph showing cell viability of HT29 colon cancer cell line when co-treated with crizotinib and XAV-939.

FIG. 5 is a graph showing cell viability of Lovo colon cancer cell line when co-treated with crizotinib and XAV-939.

FIG. 6 is a graph showing cell viability of BT-474 cell line when co-treated with L3-1Y/IgG2 and imatinib or treated with L3-1Y/IgG2 or imatinib alone.

FIG. 7 is a graph showing cell viability of BT-474 cell line when co-treated with L3-1Y/IgG2 and XAV-939 or treated with L3-1Y/IgG2 or XAV-939 alone.

FIG. 8 is a graph showing cell viability of SKBR-3 cell line when co-treated with L3-1Y/IgG2 and imatinib or treated with L3-1Y/IgG2 or imatinib alone.

FIG. 9 is a graph showing cell viability of SKBR-3 cell line when co-treated with L3-1Y/IgG2 and XAV-939 or treated with L3-1Y/IgG2 or XAV-939 alone.

FIG. 10 is a graph showing cell viability of Caki-1 cell line when co-treated with L3-1Y/IgG2 and imatinib or treated with L3-1Y/IgG2 or imatinib alone.

FIG. 11 is a graph showing cell viability of Caki-1 cell line when co-treated with L3-1Y/IgG2 and XAV-939 or treated with L3-1Y/IgG2 or XAV-939 alone.

FIG. 12 is a graph showing cell viability of L3-1Y/IgG2 resistant MKN45 cell line when co-treated with L3-1Y/IgG2 and XAV-939 or treated with L3-1Y/IgG2 or XAV-939 alone.

FIG. 13 is a graph showing cell viability of L3-1Y/IgG2 resistant EBC1 cell line when co-treated with L3-1Y/IgG2 and XAV-939 or treated with L3-1Y/IgG2 or XAV-939 alone.

FIG. 14 is a graph showing cell viability of HCC1954 cell line when co-treated with L3-1Y/IgG2 and siRNA against beta-catenin.

FIG. 15 is a graph showing cell viability of HCC1954 cell line when co-treated with crizotinib and siRNA against beta-catenin.

FIG. 16 is a graph showing cell viability of Caki-1 cell line when co-treated with L3-1Y/IgG2 and siRNA against beta-catenin.

FIG. 17 is a graph showing cell viability of Caki-1 cell line when co-treated with crizotinib and siRNA against beta-catenin.

DETAILED DESCRIPTION

c-Met inhibitors including anti-c-Met antibodies generally exhibit therapeutic effect on gastric cancer and lung cancer having high c-Met expression level, but their therapeutic effects on colon cancer and breast cancer has not been known. In the disclosure, it is revealed that the limitation of c-Met inhibitors can be overcome by combination treatment of a c-Met inhibitor with a beta-catenin inhibitor inhibiting Wnt/beta-catenin pathway (a pathway which functions to promote colon cancer and breast cancer).

In addition, even in gastric cancer and lung cancer on which a c-Met inhibitor can exhibit therapeutic effect, continuous treatment with a drug may cause a resistance thereto (acquired resistance). In this case, the treatment of a c-Met inhibitor alone may be likely to lead to side effects (agonism), and thus desired effect cannot be obtained by the single treatment. In the disclosure, it is revealed that when such resistance is induced, the resistance can be overcome by combination treatment with a beta-catenin inhibitor, thereby exhibiting desired effects.

Such combination treatment of a c-Met inhibitor and a beta-catenin inhibitor can lead to effective treatment of diseases on which a c-Met inhibitor solely has no therapeutic effect, thereby extending its indications and overcoming the resistance to the c-Met inhibitor. Similarly thereto, the limitation of beta-catenin inhibitors can also be overcome by combination treatment with a c-Met inhibitor.

The present disclosure suggests a combination therapy using a c-Met inhibitor and a beta-catenin inhibitor, which is capable of extending indications to diseases on which a c-Met inhibitor solely has no therapeutic effect, and overcoming resistance to a c-Met inhibitor or beta-catenin inhibitor. Of course, the combination therapy using a c-Met inhibitor and a beta-catenin inhibitor has more potent therapeutic effect on diseases on which a c-Met inhibitor or a beta-catenin inhibitor solely has therapeutic effect or to which no resistance is induced, by synergistic effect, and in these situations it can be possible to decrease administration dosage and/or increase administration interval, thereby decreasing side effects.

An embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating of a cancer, including a c-Met inhibitor and a beta-catenin inhibitor as active ingredients.

The pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition comprising two or more active ingredients) of a c-Met inhibitor and a beta-catenin inhibitor. The beta-catenin inhibitor and c-Met inhibitor can be present in any amount that is pharmaceutically effective when used together. The composition thus formulated can be used for simultaneous administration of the two active ingredients.

Alternatively, the c-Met inhibitor and the beta-catenin inhibitor can each be formulated in a separate composition, and the two active ingredients can be separately administered simultaneously or sequentially. For instance, a first pharmaceutical composition including a pharmaceutically effective amount of the beta-catenin inhibitor as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of the c-Met inhibitor as an active ingredient can be administered simultaneously or sequentially. In the case of the sequential administration, any order of administration may be used.

Another embodiment provides a kit useful for preventing and/or treating a cancer, including a first pharmaceutical composition including a beta-catenin inhibitor as an active ingredient, a second pharmaceutical composition including a c-Met inhibitor as an active ingredient, and a package container. The beta-catenin inhibitor and c-Met inhibitor may be used in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The package container can be any container that holds or otherwise links the two compositions in individual containers together in a single unit (e.g., a box that holds both containers, or plastic wrap that binds both containers together), or the package container may be a single, divided container having at least two chambers that each hold one of the two compositions.

The term “the pharmaceutically effective amount” as used in this specification refers to an amount of which each active ingredient can exert pharmaceutically significant effects (e.g., an amount sufficient to prevent or treat cancer in a subject).

A method of combination therapy for preventing and/or treating a cancer also is provided. The method includes co-administering a c-Met inhibitor and a beta-catenin inhibitor to a subject in need of the prevention and/or treatment of cancer. The beta-catenin inhibitor and c-Met inhibitor may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The method may further include, prior to the co-administration step, a step of identifying a subject in need of the prevention and/or treatment of cancer. The identification step may be conducted by any manners and/or methods known to relevant field for identifying whether or not a subject needs the prevention and/or treatment of cancer. For example, the step of identifying may include diagnosing a subject to have a cancer, or identifying a subject who is diagnosed as a cancer subject, particularly a cancer associated with c-Met expression.

Co-administration may be conducted by administering a mixed formulation (e.g., single composition) of a c-Met inhibitor and a beta-catenin inhibitor, as described herein. Alternatively, the c-Met inhibitor and beta-catenin inhibitor can be administered separately. The co-administration may be conducted by a first step of administering a beta-catenin inhibitor, and a second step of administering a c-Met inhibitor, wherein the first and the second administration steps may be conducted simultaneously or sequentially. In case of the sequential administration, the first step and the second step may be performed in any order, and separated by any suitable time interval (e.g., 1-60 seconds, 1-60 minutes, 1-24 hours, or 1-7 days). The beta-catenin inhibitor and c-Met inhibitor may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher.

The subject may be a mammal including a primate such as a human or a monkey, or a rodent such as a mouse or a rat, or a cell or tissue separated therefrom, or a culture of the cell or tissue.

By the co-administration of a c-Met inhibitor and a beta-catenin inhibitor, excellent and synergetic effects can be obtained as compared to the use of either single active ingredient without the other. In addition, the co-administration of a c-Met inhibitor and a beta-catenin inhibitor exhibits excellent therapeutic effect even on a disease (e.g., a cancer) on which each of a c-Met inhibitor and a beta-catenin inhibitor cannot exhibit therapeutic effect when it used alone, or a disease having resistance to each of the c-Met inhibitor and the beta-catenin inhibitor.

The “c-Met” or “c-Met proteins” refer to receptor tyrosine kinases that bind to hepatocyte growth factors (HGF). The c-Met proteins may be those derived from all kinds of species, particularly a mammal, for example, those derived from a primate such as human c-Met (e.g. NP 000236), monkey c-Met (e.g., Macaca mulatta, NP 001162100), and the like, or those derived from a rodent such as mouse c-Met (e.g., NP 032617.2), rat c-Met (e.g., NP 113705.1), and the like. These proteins may include, for example, polypeptides encoded by the nucleotide sequence identified as GenBank Accession Number NM_(—)000245, or proteins encoded by the polypeptide sequence identified as GenBank Accession Number NM_(—)000236, or extracellular domains thereof. The receptor tyrosine kinase c-Met is involved in several mechanisms including cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

Beta-catenin (or β-catenin; cadherin-associated protein) is a dual functional protein which controls cell-cell adhesion and gene transcription. Beta-catenin, one of proto-oncogenes, is present in the ubiquitinated and/or non-degraded form in various cancers and induces abnormal signal transduction. Therefore, activation of beta-catenin relates to incidence and progression of cancer. For these reasons, beta-catenin becomes important target in developing anticancer drugs. The beta-catenin may be derived from any species, and for example, beta-catenin may be one derived from a primate such as human beta-catenin (e.g., NM_(—)001098209; NP_(—)001091679), monkey beta-catenin (e.g., XM 002802839; XP 002802885), or one from a rodent such as mouse beta-catenin (e.g., NM_(—)001165902; NP_(—)001159374), rat beta-catenin (e.g., NM_(—)053357; NP_(—)445809), and the like.

The beta-catenin inhibitor which is one active ingredient of the combination therapy may be any drug targeting beta-catenin gene or beta-catenin protein. In particular, the beta-catenin inhibitor may be at least one selected from the group consisting of antibodies, aptamers, siRNA, shRNA, microRNA, inhibiting compounds (e.g., small molecular compounds or pharmaceutically acceptable salts thereof), and the like against the beta-catenin protein or gene encoding the protein. For example, the beta-catenin inhibitor may be at least one selected from the group consisting of XAV-939, imatinib, ICG-001, IWP-2, IWR-1-endo, KY02111, Wnt-059, IWR-1-exo (CAS 1127442-87-8), FH535 (CAS 108409-83-2), 1(Cardinonogen 1), CCT 031374 hydrobromide (CAS: 1219184-91-4), and pharmaceutically acceptable salts thereof (e.g., imatinib mesylate, etc.). The siRNA against beta-catenin gene may be a RNA fragment of about 5 to about 50 bp or about 10 or about 30 bp capable of binding to a gene encoding beta-catenin or a transcript thereof, and may be a siRNA including a sense strand including a nucleotide sequence of SEQ ID NOS: 113 to 127. The sequences of exemplary beta-catenin siRNAs are summarized in Table 1:

TABLE 1 Nucleotide sequence of beta-catenin siRNA sense 5′-GACUACCUGUUGUGGUUAAtt-3′ (SEQ ID NO: 113-tt) and antisense 5′- UUAACCACAACAGGUAGUCca-3′(SEQ ID NO: 128) sense 5′-CACUUGCAAUAAUUACAAAtt-3′ (SEQ ID NO: 114-tt) and antisense 5′- UUUGUAAUUAUUGCAAGUGag-3′(SEQ ID NO: 129) 5′-GAGACUGCCUUCAGAUCUU-3′ (sense) (SEQ ID NO: 115) Sense 5′-GGUGGUGGUUAAUAAGGCU-3′(SEQ ID NO: 116) and anti-sense 5′- AGCCUUAUUAACCACCACC-3′(SEQ ID NO: 130) 5′r(UGCUUGGUUCACCAGUGGAUU) (SEQ ID NO: 117) and r(GGUGUAGAACACUAAUUAA)d(TT) (SEQ ID NO: 131-d(TT)) 5′-ACAAGTAGCTGATATTGATGGACAG-3′ (sense) (SEQ ID NO: 118) 5′-GAAACGGCTTTCAGTTGAG-3′ (sense) (SEQ ID NO: 119) 5′-AAACTACTGTGGACCACAAGC-3′ (sense) (SEQ ID NO: 120) 5′-GCTTGGAATGAGACTGCTGAT-3′ (sense) (SEQ ID NO: 121) 5′-AACAGTCTTACCTGGACTCTG-3′ (sense) (SEQ ID NO: 122) 5′-AAAGGCAATCCTGAGGAAGAG-3′ (sense) (SEQ ID NO: 123) 5′-CUAUCAGGAUGACGCGG-3′ (sense) (SEQ ID NO: 124) 5′-GUCCUGUAUGAGUGGGAAC-3′ (sense) (SEQ ID NO: 125) 5′-AGCUGAUAUUGAUGGACAG-3′ (sense) (SEQ ID NO: 126) 5′-CAGGGGGUUGUGGUUAAGCUCUU-3′ (sense) (SEQ ID NO: 127)

XAV-939 is a selective Wnt/beta-catenin mediated transcription inhibitor, and has the following structure:

Imatinib (INN; Gleevac) is a tyrosine kinase inhibitor useful in treatment of various cancers, has a beta-catenin inhibiting effect, and has the following structure:

Imatinib may be used in a form of pharmaceutically acceptable salt, such as Imatinib mesylate.

The c-Met inhibitor may be any drug targeting c-Met protein or gene encoding the c-Met protein. In particular, the c-Met inhibitor may be at least one selected from the group consisting of antibodies, aptamers, siRNA, shRNA, microRNA, inhibiting compounds (e.g., small molecular compounds or pharmaceutically acceptable salts thereof), and the like against the c-Met protein or gene encoding the c-Met protein. The c-Met inhibitor may be at least one selected from the group consisting of an anti-c-Met antibody, an antigen binding fragment thereof, and a c-Met activity inhibitor.

For example, the c-Met inhibitor may be at least one selected from the group consisting of an anti-c-Met antibody or an antigen-binding fragment thereof as described below, crizotinib (PF-02341066), cabozantinib (XL-184), foretinib (E7050), PHA-665752, SU11274, SGX-523, PF-04217903, EMD 1214063, Golvatinib, INCB28060, MK-2461, tivantinib (ARQ 197), NVP-BVU972, AMG458, BMS 794833, BMS 777607, MGCD-265, AMG-208, BMS-754807, JNJ-38877605, and pharmaceutically acceptable salts thereof.

Crizotinib (PF-02341066; 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine) is one of a small molecular inhibitor against c-Met and has the following structure:

The anti-c-Met antibody or an antigen binding fragment thereof may be any type of antibody capable of specifically recognizing and/or binding to c-Met, or an antigen-binding fragment thereof. The antigen-binding fragment of the anti-c-Met antibody may be selected from the group consisting of a complementarity determining region (CDR), fragment including CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂ of the anti-c-Met antibody.

The anti-c-Met antibody may also include a variant of the antibody. The variant of the antibody may be any isotypes of antibodies derived from human and other animals and/or one including any Fc region of antibodies derived from human and other animals, having mutated hinge wherein at least one amino acid is changed, deleted or added. Unless stated otherwise, the anti-c-Met antibody may include the variants of the antibody as well as the antibody with no variation.

In a particular embodiment, the anti-c-Met antibody may recognize a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope. It may be any antibody or antigen-binding fragment that acts on c-Met to induce c-Met intracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor, may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region including the amino acid sequence of SEQ ID NO: 71, which corresponds to amino acids 106 to 124 of the SEMA domain (SEQ ID NO: 79), is a loop region between the second and the third beta propellers within the epitopes of the SEMA domain. It may act as an epitope for the anti-c-Met antibody of the present disclosure.

The term “epitope,” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more consecutive or non-consecutive amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 consecutive or non-consecutive amino acid residues within the amino acid sequence of SEQ ID NO: 71 which corresponds to a range from residue 106 to residue 124 within the SEMA domain (SEQ ID NO: 79) of a c-Met protein. For example, the epitope may be a polypeptide having 5 to 19 consecutive amino acids of the amino acid sequence of SEQ ID NO: 71, which sequence includes the amino acid sub-sequence EEPSQ (SEQ ID NO: 73) that serves as an essential element for the epitope. For example, the epitope may be a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third beta propellers within the SEMA domain of a c-Met protein, and the epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or an antigen-binding fragment according to one embodiment of the present disclosure most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 consecutive amino acids of the amino acid sequence of SEQ ID NO: 71, which consecutive amino acids include SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may include:

at least one heavy chain complementarity determining region (CDR) selected from the group consisting of CDR-H1 including the amino acid sequence of SEQ ID NO: 4; CDR-H2 including the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2, or including an amino acid sequence of 8 to 19 consecutive amino acids within SEQ ID NO: 2 including amino acid residues from 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and CDR-H3 including the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 85, or including an amino acid sequence of 6 to 13 consecutive amino acids within SEQ ID NO: 85 including amino acid residues from 1^(st) to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region (CDR) selected from the group consisting of CDR-L1 including the amino acid sequence of SEQ ID NO: 7, CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and CDR-L3 including the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15 or SEQ ID NO: 86 or SEQ ID NO: 89, or including an amino acid sequence of 9 to 17 consecutive amino acids within SEQ ID NO: 89 including amino acid residues from 1^(st) to 9^(th) positions of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are represented, respectively by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn- Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 27, 28, and 85. The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may include

a heavy variable region including a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 27, 28, and 85;

a light variable region including a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOs 12, 13, 14, 15, 16, 37, 86, and 89; or

a combination of the heavy variable region and the light variable region.

In one embodiment, the anti-c-Met antibody or antigen-binding fragment may include a heavy chain variable region including an amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and a light chain variable region including an amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99 or 107.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced from a hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein

(See Korean Patent Publication No. 2011-0047698, the disclosure of which is incorporated in its entirety herein by reference).

The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62; the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide) or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64; and the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide) or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68; the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide) or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

The anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

In particular embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68.

The polypeptide with the amino acid sequence of SEQ ID NO: 70 is a light chain including human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by such replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 of SEQ ID NO: 108 (corresponding to position 52 of SEQ ID NO: 68, which corresponds to position 27e according to kabat numbering; positioned within CDR-L1) of the polypeptide with the amino acid sequence through 21^(st) to 240^(th) positions of SEQ ID NO: 68 with tryptophan. By such replacement, antibodies and antibody fragments including said sequences exhibit increased activities compared to an unmodified sequence of the same type, such as increased c-Met biding affinity, c-Met degradation activity, Akt phosphorylation activity, and the like.

In another embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence of SEQ ID NO: 109 or 111 and a light chain including SEQ ID NO: 110 or 112.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies are developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDR of animal-derived antibodies. Antibody database, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti-c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies or human antibodies. The anti-c-Met antibodies may be monoclonal. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be synthetic or recombinant.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” or “specifically recognized” is well known to one of ordinary skill in the art, and indicates that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, or 105. Preferably, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

The remaining portions of the antibody other than the CDR region, the heavy chain variable region, or the light chain variable region, for example, a heavy chain constant region and/or a light chain constant region, may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).

In one embodiment, the antibody may be an antigen-binding fragment selected from the group consisting of scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂.

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. For example, the antigen-binding fragment may be scFv, (scFv)₂, Fab, Fab′, or F(ab′)₂, but is not limited thereto. Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(m), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be the same as described in the above, for example, those having the amino acid length of about 1 to about 100, about 2 to about 50, particularly about 5 to about 25, and any kinds of amino acids may be included without any restriction.

The antigen-binding fragments may be obtained using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

The mixed formulation including an effective amount of the beta-catenin inhibitor and an effective amount of the c-Met inhibitor, the first pharmaceutical composition including an effective amount of the beta-catenin inhibitor as an active ingredient, or the second pharmaceutical composition including an effective amount of the c-Met inhibitor as an active ingredient may be provided optionally along with a pharmaceutically acceptable carrier, diluent, and/or excipient.

The pharmaceutically acceptable carriers that may be included in the mixed formulation or the pharmaceutical compositions may be those commonly used in formulations of drugs, and may be, but not limited to, at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. Besides these components, the mixed formulation or the pharmaceutical compositions may further include at least one selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and a preservative.

The mixed formulation or the pharmaceutical compositions may be administered orally or parenterally. Parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

The pharmaceutically effective amount of the beta-catenin inhibitor and the pharmaceutically effective amount of the c-Met inhibitor for a single dose may be prescribed in a variety of ways, depending on factors such as formulation methods, administration manners, age of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the pharmaceutically effective amount of the beta-catenin inhibitor for a single dose may be in ranges of about 0.001 to about 100 mg/kg, or about 0.02 to about 10 mg/kg, and the pharmaceutically effective amount of the c-Met inhibitor for a single dose may be in ranges of about 0.001 to about 100 mg/kg, or about 0.02 to about 10 mg/kg, but not limited thereto.

The pharmaceutically effective amount for the single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. For the kit, the pharmaceutically effective amount of the beta-catenin inhibitor and the pharmaceutically effective amount of the c-Met inhibitor for the single dose (one-time administration) may be each contained in a package container as a base unit.

The administration interval between the co-administrations is defined as a period between the co-administration and the subsequent co-administration may be, but not limited to, about 24 hours to about 30 days and particularly about 7 to about 14 days or so. In case that the co-administration comprises the sequential performance of the first administration step of administering the pharmaceutically effective amount of the beta-catenin inhibitor and the second administration step of administering the effective amount of the c-Met inhibitor, the administration interval between the first administration step and the second administration step may be simultaneous, or about 1 second to about 60 min., 1-24 hours, or 1-7 days, particularly about 1 to about 10 min., and their administration may occur in any order.

The mixed formulation or the pharmaceutical compositions for co-administration may be a solution in oil or an aqueous medium, a suspension, a syrup, or an emulsifying solution form, or they may be formulated into a form of an extract, powders, granules, a tablet or a capsule, and they may further include a dispersing agent or a stabilizing agent for their formulation.

In embodiments where the c-Met inhibitor is an anti-c-Met antibody or an antigen binding fragment thereof, the pharmaceutically effective amount of the c-Met inhibitor as an active ingredient may be formulated into an immunoliposome. A liposome containing an antibody may be prepared using any methods well known in the pertinent field. The immunoliposome is a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivated phosphatidylethanolamine, which may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may further be included in the liposome.

The pharmaceutical composition and method for co-administration proposed in this disclosure can be used for preventing and/or treating a cancer. The cancer may be related to overexpression and/or abnormal activation of c-Met and/or beta-catenin. The cancer may be a solid cancer or blood cancer. The cancer may be a cancer on which a c-Met inhibitor or a beta-catenin inhibitor has no anticancer effect when treated alone or which has resistance to the c-Met inhibitor or beta-catenin inhibitor, as well as a cancer on which the c-Met inhibitor or beta-catenin inhibitor has anticancer effect when treated alone or which has no resistance to the c-Met inhibitor or beta-catenin inhibitor. For instance, the cancer may be, not limited to, at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, osteosarcoma and so on. In a particular embodiment, the cancer may be a cancer on which a c-Met inhibitor has no anticancer effect (for example, a c-Met inhibitor-resistant cancer), and may be selected from the group consisting of as a colon cancer, a breast cancer, a kidney cancer, and the like. The cancer may include a metastatic cancer as well as a primary cancer.

The prevention and/or treatment effects of the cancer may include effects of not only inhibiting the growth of the cancer cells but also inhibiting the ability of the cancer to migrate, invade healthy cells, and metastasize.

In another embodiment, a combination therapy capable of overcoming resistance to each other and improving therapeutic effect is provided. In particular, a beta-catenin inhibitor is suggested as a partner for combination therapy using a c-Met inhibitor, which can lead to exhibiting therapeutic effect on a disease on which a c-Met inhibitor has no therapeutic effect when treated alone or which has resistance to the c-Met inhibitor. Alternatively, a c-Met inhibitor is suggested as a partner for combination therapy using a beta-catenin inhibitor, which can lead to exhibiting therapeutic effect on a disease on which a beta-catenin inhibitor has no therapeutic effect when treated alone or which has resistance to the beta-catenin inhibitor.

Another embodiment provides a pharmaceutical composition for improving the efficacy of a c-Met inhibitor, where the pharmaceutical composition includes a beta-catenin inhibitor. Another embodiment provides a method for improving the efficacy of a c-Met inhibitor, including administering a beta-catenin inhibitor together with the c-Met inhibitor to a subject in need thereof. Another embodiment provides a use of a beta-catenin inhibitor for improving the efficacy of a c-Met inhibitor.

Another embodiment provides a pharmaceutical composition for improving the efficacy of a beta-catenin inhibitor, where the pharmaceutical composition includes a c-Met inhibitor. Another embodiment provides a method for improving the efficacy of a beta-catenin inhibitor, including administering a c-Met inhibitor together with the beta-catenin inhibitor to a subject in need thereof. Another embodiment provides a use of a c-Met inhibitor for improving the efficacy of a beta-catenin inhibitor.

The improvement of efficacy of the c-Met inhibitor or the beta-catenin inhibitor may include that the inhibitor possesses a therapeutic effect on a disease (e.g., a cancer) on which the inhibitor has no therapeutic effect when used alone or has an effect only on a disease (e.g., a cancer) which has resistance to each inhibitor when used alone.

EXAMPLES

One or more embodiments of the present invention will now be described in further detail with reference to the following Examples. However, these examples are for the illustrative purposes only and are not intended to limit the scope of the invention.

Reference Example Construction of Anti-c-Met Antibody Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (See Korean Patent Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with a filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Experimental Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has an identity/identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S-*T), 48 (V-*L), 73 (D-*N), and 78 (T-*L). Then, H1 was further mutated at positions 83 (R-*K) and 84 (A-*T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41). For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has an identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have an identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. One day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 2, below.

TABLE 2 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 3 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 3 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™ SFM (Invitrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was representatively selected for the following examples, and referred as L3-1Y-IgG2.

Example 1 Effect of Co-Administration of an Anti-c-Met Antibody and a Beta-Catenin Inhibitor (XAV-939) in Breast Cancer Cell

To confirm the effect of co-administration in a breast cancer cell on which an anti-c-Met antibody has no effect when administered alone, HCC1954 (ATCC, CRL 2338) cells were used for assaying the effect of co-administration on cell proliferation.

In particular, 5000 cells of HCC1954 cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). 24 hours after, the incubated cells were treated with L3-1Y/IgG2 prepared in reference example and a beta-catenin inhibitor XAV-939 (Selleck chemical). In particular, 72 hours after the antibody and inhibitor treatment, 100 μL (microliter) of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer). In the experiment, L3-1Y/IgG2 was used at the amount of 0, 0.016, 0.08, 0.4, and 2 μg/ml, and XAV-939 was used at the fixed amount of 10 μM (micromole).

The obtained result is demonstrated in FIG. 1. As shown in FIG. 1, in HCC1954 cells, when L3-1Y/IgG2 is administered alone, no cell proliferation inhibition effect is observed, and rather, the cell proliferation level is increased with increasing concentration of L3-1Y/IgG2. In contrast, when L3-1Y/IgG2 and XAV-939 are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of L3-1Y/IgG2. These results indicate that the co-administration of L3-1Y/IgG2 and XAV-939 can lead to extending the indication on which L3-1Y/IgG2 has cancer cell proliferation inhibition effect to a breast cancer (i.e., L3-1Y/IgG2 and XAV-939 inhibits the effects of breast cancer cell proliferation).

Example 2 Effect of Co-Administration of an Anti-c-Met Antibody and a Beta-Catenin Inhibitor (Imatinib Mesylate) in Breast Cancer Cell

To confirm the effect of co-administration in a breast cancer cell on which an anti-c-Met antibody has no effect when administered alone, HCC1954 (ATCC, CRL 2338) cells were used for assaying the effect of co-administration on cell proliferation.

In particular, 5000 cells of HCC1954 cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). 24 hours after, the incubated cells were treated with L3-1Y/IgG2 prepared in reference example and a beta-catenin inhibitor Imatinib mesylate (Gleevac, Novartis; hereinafter, referred as ‘imatinib’). In particular, 72 hours after the antibody treatment, 100 μL of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer). In the experiment, L3-1Y/IgG2 was used at the amount of 0, 0.016, 0.08, 0.4, and 2 μg/ml, and imitanib was used at the fixed amount of 10 uM. Imitanib (Gleevac) targets Bcr-Abl enzyme which activates β-catenin, and thus Imitanib can directly inhibit β-catenin.

The obtained result is demonstrated in FIG. 2. As shown in FIG. 2, in HCC1954 cells, when L3-1Y/IgG2 is administered alone, no cell proliferation inhibition effect is observed, and rather, the cell proliferation level is increased with increasing concentration of L3-1Y/IgG2. In contrast, when L3-1Y/IgG2 and imitanib are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of L3-1Y/IgG2. These results indicate that the co-administration of L3-1Y/IgG2 and imitanib can lead to extending the indication on which L3-1Y/IgG2 has cancer cell proliferation inhibition effect to a breast cancer (i.e., L3-1Y/IgG2 and imitanib inhibits the effects of breast cancer cell proliferation).

Example 3 Effect of Co-Administration of an Anti-c-Met Antibody and a Beta-Catenin Inhibitor in Colon Cancer Cell

To confirm the effect of co-administration in a colon cancer cell on which an anti-c-Met antibody has no effect when administered alone, HT-29 (ATCC, HTB-38) cells were used for assaying the effect of co-administration on cell proliferation.

In particular, 5000 cells of HT-29 cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). 24 hours after, the incubated cells were treated with L3-1Y/IgG2 prepared in reference example or anti-c-Met antibody 1 (heavy chain: SEQ ID NO: 109; light chain: SEQ ID NO: 110) and beta-catenin inhibitor XAV-939 (Selleck chemical). In particular, 72 hours after the antibody treatment, 100 uL of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer). In the experiment, L3-1Y/IgG2 was used at the amount of 0, 0.016, 0.08, 0.4, and 2 μg/ml, and XAV-939 was used at the fixed amount of 5 uM.

The obtained result is demonstrated in FIG. 3A. As shown in FIG. 3A, in HT-29 cells, when L3-1Y/IgG2 or anti-c-Met antibody 1 is administered alone, no cell proliferation inhibition effect is observed. In contrast, when L3-1Y/IgG2 or anti-c-Met antibody 1 and XAV-939 are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of L3-1Y/IgG2 or anti-c-Met antibody 1. These results indicate that the co-administration of L3-1Y/IgG2 or anti-c-Met antibody 1 and XAV-939 can lead to extending the indication on which L3-1Y/IgG2 or anti-c-Met antibody 1 has cancer cell proliferation inhibition effect to a colon cancer (i.e., L3-1Y/IgG2 or anti-c-Met antibody 1 and XAV-939 inhibits the effects of colon cancer cell proliferation).

In addition, 5000 cells of HT-29 cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). The provided cells were treated with 2 μg/ml of L3-1Y/IgG2 and 10 uM of Imatinib mesylate (Gleevac, Novartis; hereinafter, referred as ‘imatinib’) alone or in combination. The obtained cell viability is demonstrated in FIG. 3B. As shown in FIG. 3B, the co-administration of L3-1Y/IgG2 and imatinib leads to considerably increased colon cancer cell death rate, compared to administration of L3-1Y/IgG2 or imatinib alone.

Example 4 Effect of Co-Administration of a c-Met Inhibitor (Crizotinib) and a Beta-Catenin Inhibitor in Colon Cancer Cell (HT-29)

To confirm the effect of co-administration in a colon cancer cell on which a c-Met inhibitor has no effect when administered alone, HT-29 (ATCC, HTB-38) cells were used for assaying the effect of co-administration on cell proliferation.

In particular, 5000 cells of HT-29 cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). 24 hours after, the incubated cells were treated with a small molecular compound inhibiting c-Met, crizotinib (Selleck chemical) and beta-catenin inhibitor XAV-939 (Selleck chemical). In particular, 72 hours after the antibody treatment, 100 uL of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer). In the experiment, crizotinib was used at the amount of 0, 0.16, 0.8, 4, and 20 nM, and XAV-939 was used at the fixed amount of 2 uM.

The obtained result is demonstrated in FIG. 4. As shown in FIG. 4, in HT-29 cells, when crizotinib is administered alone, no cell proliferation inhibition effect is observed. In contrast, when crizotinib and XAV-939 are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of crizotinib. These results indicate that the co-administration of crizotinib and XAV-939 can lead to extending the indication on which crizotinib has cancer cell proliferation inhibition effect to a colon cancer (i.e., crizotinib and XAV-939 inhibits the effects of colon cancer cell proliferation).

Example 5 Effect of Co-Administration of a c-Met Inhibitor (Crizotinib) and a Beta-Catenin Inhibitor in Colon Cancer Cell (Lovo)

To confirm the effect of co-administration in a colon cancer cell on which a c-Met inhibitor has no effect when administered alone, Lovo (ATCC, CCL-229) cells were used for assaying the effect of co-administration on cell proliferation.

In particular, 5000 cells of Lovo cell were incubated in 96-well plate (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). 24 hours after, the incubated cells were treated with a small molecular compound inhibiting c-Met, crizotinib (Selleck chemical) and beta-catenin inhibitor XAV-939 (Selleck chemical). In particular, 72 hours after the antibody treatment, 100 uL of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer). In the experiment, L3-1Y/IgG2 was used at the amount of 0, 0.016, 0.08, 0.4, and 2 μg/ml, and XAV-939 was used at the fixed amount of 5 uM.

The obtained result is demonstrated in FIG. 5. As shown in FIG. 5, in HT-29 cells, when crizotinib is administered alone, no cell proliferation inhibition effect is observed. In contrast, when crizotinib and XAV-939 are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of crizotinib. These results indicate that the co-administration of crizotinib and XAV-939 can lead to extending the indication on which crizotinib has cancer cell proliferation inhibition effect to a colon cancer (i.e., crizotinib and XAV-939 inhibits the effects of colon cancer cell proliferation).

Example 6 Effect of Co-Administration of an Anti-c-Met Antibody and a Beta-Catenin Inhibitor in Cells on which the Anti-c-Met Antibody Solely has No Effect

To confirm the effect of co-administration in a colon cancer cell on which an anti-c-Met antibody has no effect when administered alone, a breast cancer cell line (BT-474, SKBR-3; obtained from ATCC) and a kidney cancer cell line (Caki-1; ATCC) were used for assaying the effect of co-administration of an anti-c-Met antibody and a beta-catenin inhibitor on cell proliferation.

5000 cells of each cell line were added to 96-well plate and incubated in RPMI 1640 medium (GIBCO; including 10% FBS) at 37° C.

Referring to the method described in Examples 1 to 3, the incubated cells were treated with L3-1Y/IgG2 2 μg/ml and a beta-catenin inhibitor Imatinib mesylate (Gleevac, Novartis; hereinafter, referred as ‘imatinib’) 10 μM or 0.4 μM, or XAV-939 2 μM, 5 μM, or 10 μM alone or in combination, and the cell viability was measured.

The obtained results are demonstrated in FIGS. 6 to 11. FIG. 6 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 10 μM of imatinib in BT-474 cell line. FIG. 7 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 5 uM of XAV-939 in BT-474 cell line. FIG. 8 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 10 μM of imitanib in SKBR-3 cell line. FIG. 9 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 10 μM of XAV-939 in SKBR-3 cell line. FIG. 10 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 0.4 μM of imatinib in Caki-1 cell line. FIG. 11 shows the results of co-administration or single-administration of 2 μg/ml of L3-1Y/IgG2 and 2 uM of XAV-939 in Caki-1 cell line. As known from these results, on cancer cell lines on which L3-1Y/IgG2 has no cell proliferation inhibition effect when administered alone, the co-administration of L3-1Y/IgG2 and a beta-catenin inhibitor leads to considerably increased cancer cell proliferation inhibition effect compared to single administration.

Example 7 Effect of Co-Administration of an Anti-c-Met Antibody and a Beta-Catenin Inhibitor in Anti-c-Met Antibody Resistant Cells

To prepare anti-c-Met antibody resistant clones, MKN45 cell (JCRB, JCRB0254) and EBC1 cell (JCRB, JCRB0820) were exposed to L3-1Y/IgG2 in vitro for 3 months or more. During the period, L3-1Y/IgG2 was treated at increased amount from 1 μg/ml to 10 μg/ml. To confirm the generation of acquired resistance to L3-1Y/IgG2, the survival or death of the cells was tested by culturing the cells under the presence or absence of L3-1Y/IgG2 for 5 weeks, and survived cells under the presence of L3-1Y/IgG2 were collected.

Referring to the method of Examples 1 to 3, in the obtained L3-1Y/IgG2 resistant cells, the effect the co-administration of L3-1Y/IgG2 and a beta-catenin inhibitor XAV-939 was tested. L3-1Y/IgG2 was used at the concentration of 0, 0.016, 0.08, 0.4, or 2 μg/ml, and XAV-939 was used at the fixed concentration of 2 uM.

The obtained results are demonstrated in FIG. 12 (L3-1Y/IgG2 resistant MKN45 cell; MKN45-re) and FIG. 13(L3-1Y/IgG2 resistant EBC1 cell; EBC1-RE). As shown in FIGS. 12 and 13, the co-administration of L3-1Y/IgG2 and a beta-catenin inhibitor can lead to considerably increased cancer cell proliferation inhibition effect even in L3-1Y/IgG2 resistant cell, compared to single administration of L3-1Y/IgG2.

Example 8 Effect of Co-Administration of a c-Met Inhibitor and Beta-Catenin siRNA in Cells on which the Anti-c-Met Antibody Solely has No Effect

To confirm the effect of co-administration of c-Met inhibitor (anti-c-Met antibody L3-1Y/IgG2 or crizotinib) and beta-catenin siRNA in a cancer cell on which a c-Met inhibitor has no effect when administered alone, a breast cancer cell line HCC1954 (ATCC, CRL 2338) and a kidney cancer cell line Caki-1 (ATCC) were used for assaying the effect of the co-administration.

In particular, in 96-well plate, beta-catenin siRNA (Dharmacon, SMARTpool) was subjected to reverse transfection into 5000 cells/well of HCC1954 cell or Caki-1 cell and the cells were incubated (medium: 10% FBS in RPMI 1640 (GIBCO), incubation temperature: 37° C.). The reverse transfection was performed using lipofectamine RNAi max (invitrogen0. The reverse transfection was performed by pre-incubating 10˜20 uM of siRNA diluted in opti-MEM (Gibco) and lipofectamine RNAi max diluted in opti-MEM (Gibco) at room temperature for 15 minutes, and then mixing with 5000 cells/well of each cell, to allow reverse-transfection. 24 hours after, the prepared cells were treated with L3-1Y/IgG2 at the concentration of 0, 0.016, 0.08, 0.4, and 2 μg/ml, and crizotinib (Selleck chemical) at the concentration of 0, 0.16, 0.8, 4, 20 nM. In particular, 72 hours after the antibody treatment, 100 uL of CellTiter Glo solution (Promega, G7572) was added to each well and left at room temperature for 30 minutes. The number of the cells was counted through luminescence signal, and the luminescence signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer).

The obtained results are demonstrated in FIG. 14 (co-treatment of L3-1Y/IgG2 and beta-catenin siRNA in HCC1954 cells), FIG. 15 (co-treatment of crizotinib and beta-catenin siRNA in HCC1954 cells), FIG. 16 (co-treatment of L3-1Y/IgG2 and beta-catenin siRNA in Caki-1 cells), and FIG. 17 (co-treatment of crizotinib and beta-catenin siRNA in Caki-1 cells). As shown in FIGS. 14 to 17, in HCC1954 cells or Caki-1 cells, when L3-1Y/IgG2 or crizotinib is treated alone, no cell proliferation inhibition effect is observed, and rather, the cell proliferation level is increased with increasing concentration of L3-1Y/IgG2 or crizotinib. In contrast, when L3-1Y/IgG2 or crizotinib and beta-catenin siRNA (represented by “sib-catenin”) are co-administered, an inhibition effect on cell proliferation is clearly observed, and such inhibition effect depends on the concentration of L3-1Y/IgG2 or crizotinib. These results indicate that the co-administration of a c-Met inhibitor and beta-catenin siRNA can lead to extending the indication on which a c-Met inhibitor, in particular L3-1Y/IgG2, has cancer cell proliferation inhibition effect to a breast cancer or a kidney cancer.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method of combination therapy for preventing or treating a cancer comprising co-administering a c-Met inhibitor and a beta-catenin inhibitor to a subject in need thereof.
 2. The method of claim 1, wherein the c-Met inhibitor comprises at least one selected from the group consisting of an anti-c-Met antibody or an antigen-binding fragment thereof, an aptamer, siRNA, shRNA, microRNA, a small molecule c-Met inhibitor against c-Met protein or a gene encoding the c-Met protein, pharmaceutically acceptable salts thereof, and combinations thereof.
 3. The method of claim 2, wherein the c-Met inhibitor comprises an anti-c-Met antibody or antigen binding fragment thereof, and the anti-c-Met antibody or antigen binding fragment thereof specifically binds to an epitope comprising 5 to 19 consecutive amino acids of SEQ ID NO: 71 including SEQ ID NO:
 73. 4. The method of claim 2, wherein c-Met inhibitor comprises an anti-c-Met antibody or antigen binding fragment thereof, and the anti-c-Met antibody or an antigen-binding fragment thereof comprises: at least one heavy chain complementarity determining region (CDR) selected from the group consisting of a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2, or comprising an amino acid sequence of 8 to 19 consecutive amino acids within SEQ ID NO: 2 including the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or comprising an amino acid sequence of 6 to 13 consecutive amino acids within SEQ ID NO: 85 including the 1^(st) to 6^(th) positions of SEQ ID NO: 85; at least one light chain complementarity determining region (CDR) selected from the group consisting of a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, SEQ ID NO: 89, or 9 to 17 consecutive amino acids within SEQ ID NO: 89 including the 1^(st) to 9^(th) positions of SEQ ID NO: 89; or a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region.
 5. The method of claim 4, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 22, 23, and 24, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 25, and 26, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 27, 28, and 85; a light chain variable region comprising a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 29, 30, 31, 32, 33, and 106, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 34, 35, and 36, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 37, 86, and 89; or a combination thereof.
 6. The method of claim 4, wherein anti-c-Met antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99 or 107, or a combination thereof.
 7. The method of claim 4, wherein the anti-c-Met antibody comprises: a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, the amino acid sequence of the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of the 18^(th) to 461^(st) positions of SEQ ID NO: 64, and the amino acid sequence of the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 108, the amino acid sequence of the 21^(st) to 240^(th) positions of SEQ ID NO: 68, and the amino acid sequence of the 21^(st) to 240^(th) positions of SEQ ID NO:
 70. 8. The method of claim 7, wherein the anti-c-Met antibody comprises a heavy chain comprising the amino acid sequence of the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising the amino acid sequence of the 21^(st) to 240^(th) positions of SEQ ID NO:
 68. 9. The method of claim 2, wherein the c-Met inhibitor comprises an anti-c-Met antibody or antigen binding fragment thereof, and the anti-c-Met antibody comprises a heavy chain comprising SEQ ID NO: 109 or 111; and a light chain comprising SEQ ID NO: 110 or
 112. 10. The method of claim 2, wherein the c-Met inhibitor comprises crizotinib, cabozantinib, foretinib, PHA-665752, SU11274, SGX-523, PF-04217903, EMD 1214063, Golvatinib, INCB28060, MK-2461, tivantinib, NVP-BVU972, AMG458, BMS 794833, BMS 777607, MGCD-265, AMG-208, BMS-754807, JNJ-38877605, or a pharmaceutically acceptable salt thereof.
 11. The method of claim 1, wherein the beta-catenin inhibitor comprises an anti-beta-catenin antibody or an antigen-binding fragment thereof an aptamer, siRNA, shRNA, microRNA, or small molecule inhibitor of beta-catenin protein or a gene encoding the beta-catenin protein; a pharmaceutically acceptable salt thereof or a combination thereof.
 12. The method of claim 11, wherein the beta-catenin inhibitor comprises XAV-939, imatinib, ICG-001, IWP-2, IWR-1-endo, KY02111, Wnt-059, IWR-1-exo, FH535, Cardinonogen 1, CCT 031374 hydrobromide, or a pharmaceutically acceptable salt thereof or siRNA comprising a sense strand comprising one of SEQ ID NOs: 113 to
 127. 13. The method of claim 1, wherein the cancer is an anti-c-Met inhibitor-resistant cancer.
 14. The method of claim 1, wherein the cancer is squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, or osteosarcoma.
 15. The method of claim 14, wherein the cancer is colon cancer, breast cancer, or kidney cancer.
 16. A method for improving the efficacy of a c-Met inhibitor, comprising administering a beta-catenin inhibitor together with the c-Met inhibitor to a subject.
 17. A method for improving the efficacy of a beta-catenin inhibitor, comprising administering a c-Met inhibitor together with the beta-catenin inhibitor to a subject.
 18. A pharmaceutical composition comprising a c-Met inhibitor and a beta-catenin inhibitor.
 19. A kit comprising a first pharmaceutical composition comprising a pharmaceutically effective amount of a c-Met inhibitor as an active ingredient, a second pharmaceutical composition comprising a pharmaceutically effective amount of a beta-catenin inhibitor as an active ingredient, and a package container containing the first and second pharmaceutical compositions. 